The bacterium Escherichia coli ( or
E. coli ) is an ideal organism for the molecular geneticist to
manipulate and has been used extensively in recombinant DNA research.
It is a common inhabitant of the human colon and can easily be grown
in suspension culture in a nutrient medium such as Luria broth, or in
a petri dish of Luria broth mixed with agar (LB agar) or nutrient
agar.

The single circular chromosome of E. coli contains about
five million DNA base pairs, only 1/600th the haploid amount of DNA
in a human cell. In addition, the E.coli cell may contain
small circular DNA molecules (1,000 to 200,000 base pairs) called
plasmids, which also carry genetic information. The plasmids
are extra chromosomal; they exist separately from the chromosome.
Some plasmids replicate only when the bacterial chromosome
replicates, and usually exists only as single copies within the
bacterial cell. Others replicate autonomously and often occur in as
many as 10 to 200 copies within a single bacterial cell. Certain
plasmids, called R plasmids, carry genes for resistance to
antibiotics such as ampicillin, kanamycin, or tetracycline.

In nature, genes can be transferred between bacteria in three
ways: conjugation, transduction, and transformation.
Conjugation is a mating process during which genetic material
is transferred from one bacterium to another of a different mating
type. Transduction requires the presence of a virus to act as
a vector to transfer small pieces of DNA from one bacterium to
another. Bacterial transformation involves transfer of genetic
information into a cell by direct uptake of the DNA. During gene
transfer, the uptake and expression of foreign DNA by recipient
bacterium can result in the conferring a particular trait to a
recipient lacking the trait.

Plasmids can transfer genes that occur naturally within them, or
plasmids can act as carriers for introducing foreign DNA from other
bacteria, plasmids, or even eukaryotes into recipient bacterial
cells. Restriction endonucleases can be used to cut and insert pieces
of foreign DNA into the plasmid vectors (figure 6.1).

Figure 6.1 Bacterial Transformation using a Restriction
Endonuclease

Exercise 6A: Bacterial Transformation-Ampicillin
Resistance*

Background Information

You will insert a plasmid that contains a gene for the resistance
to ampicillin , an antibiotic that is lethal to many bacteria, into
competent E.coli cells. Transformed bacteria can be selected
based on their resistance to ampicillin by spreading the transformed
cells on nutrient medium containing ampicillin. Any cell that grown
on this mediums has been transformed.

Procedure

1. Mark one 15 mL tube "+"; this tube will have the plasmid added
to it. Mark another tube "-" ; this tube will have no plasmid
added.

2. Use a sterile pipette to add 250 micro liters (uL) of ice
cold 0.05M CaCl2 to each tube.

3. Transfer a large (3 mm) colony of E.coli from a starter plate
to each of the tubes using using a sterile inoculating loop.
Try and get the same amount of bacteria into each tube. Be careful
not to transfer any agar.

4. Vigorously tap the loop against the wall of the tube to
dislodge the cell mass.

5. Mix the suspension by repeatedly drawing in and emptying a
sterile micro pipette with the suspension.

8. While the tubes are on ice, obtain two LB agar plates and two
LB/Amp agar(LB agar containing ampicillin) plates. Label each plate
on the bottom as follows: one LB agar plate "LB+" and the other
"LB-". Label one LB/Amp plate "LB/Amp+" and the other "LB/Amp-."

9. A brief pulse of heat facilitates entry of foreign DNA into the
E. coli cells. Heat shock cells in both the "+" and "-" tubes
by holding the tubes in a 42 degree C water bath for 90 seconds. It
is essential that cells be given a sharp and distinct shock, so take
the tubes directly from the ice to the 42 degree C water bath.

10. Immediately return cells to ice for two minutes.

11. Use sterile micro pipette to add 250 uL of Luria broth to each
tube. Mix by tapping with your finger and set at room temperature.
Any transformed cells are now resistant to ampicillin because they
possess the gene whose product renders the antibiotic
ineffective.

12. Place 100 uL of "+" cells on the "LB+" plate and on the
"LB/Amp+" plate. Place 100 uL of "-" cells on the "LB-" plate and on
the "LB/Amp-" plate.

13. Immediately spread the cells using a sterile spreading rod. (
Remove the spreading rod from alcohol and briefly pass it through a
flame. Cool by touching it to the agar on a part of the dish away
from the bacteria. Spread the cells and once again immerse the rod in
alcohol and flame it.) Repeat the procedure for each plate.

14. Allow plates to set for several minutes. Tape your plates
together and incubate inverted overnight at 37 degrees C.

* Exercise 6A is adapted with permission from DNA
Science: A First Course in Recombinant-DNA Technology by David A
Micklos, DNA Learning center of Cold Spring Harbor Laboratory , and
Greg A. Freyer, Columbia University College of Physicians and
Surgeons, Copyright 1990 Cold Spring Harbor Laboratory Press and
Carolina Biological Supply Company. It is based on a protocol
published by Douglas Hanahan, University of California, San
Francisco

Analysis of Results

1. Observe the colonies through the bottom of the culture plate.
Do not open the plates. Count the number of individual
colonies; use a permanent marker to mark each colony as it is
counted. If cell growth is too dense to count individual colonies,
record "lawn."

3. Transformation efficiency is expressed as the number of
antibiotic-resistant colonies per microgram of pAMP. Because
transformation is limited to only those cells that are competent,
increasing the amount of plasmid used does not necessarily increase
the probability that a cell will be transformed. A sample of
competent cells is usually saturated with small amounts of plasmid
and excess DNA may actually interfere with the transformation
process.

a. Determine the total mass of pAMP used.
_____________________

( you used 10 uL of pAMP at a concentration of 0.005ug/uL.)

Total Mass = volume x concentration.

b. Calculate the total volume of cell suspension prepared.
_______________________

c. Now calculate the fraction of the total cell suspension that
was spread on the plate.

Restriction enzymes or restriction endonucleases are essential
tools in recombinant DNA methodology. Several hundred have been
isolated from a variety of prokaryotic organisms. Restriction
endonucleases are named according to a specific system of
nomenclature. The letters refer to the organism from which the enzyme
was isolated. The first letter of the name stands for the genus name
of the organism. The next two letters represent the second word or
the species name. The fourth letter (if there is one) represents the
strain of the organism. Roman numerals indicate whether the
particular enzyme was the first isolated, the second, or so on.

Examples:fffffffffffffffffffffffffffffffffffffff

EcoRI E = genus Escherichia

cco= species coli

cR = strain RY13

ccccccccccccccI = first endonuclease
isolated

HaeII H = genus Haemophilus

vfcae= species aegyptus

ccccccccccccccvII I = second
endonuclease isolated

Restriction endonucleases recognize specific DNA sequences in
double stranded DNA (usually a four to six base pair sequence of
nucleotides) and digest the DNA at these sites. The result is the
production of fragments of DNA of DNA of various lengths. Some
restriction enzymes cut cleanly through helix at the same position on
both strands to produce fragments with blunt ends ( figure 6.2a ).
Other endonucleases cleave each strand off center at specific
nucleotides to produce fragments with "overhangs" or sticky ends
(figure 6.2b). By using the same restriction enzyme to "cut" DNA from
two different organisms, complementary "overhangs" or sticky ends
will be produced and allow the DNA from two sources to be
"recombined."

Figure 6.2a

Hae III

Cleavage by HaeIII produces blunt
ends

5'...GGCC...3'

3'...CCGG...5'

Figure 6.2b

EcoR I

Cleavage by EcoRI produces sticky
ends

5'...GAATTC...3'

3'...CTTAAG...5'

In this exercise, samples of DNA obtained from the
bacteriophage lambda have been incubated with different
restriction enzymes. The resulting fragments of DNA will be separated
by using gel electrophoresis. One sample has been digested with the
restriction endonuclease EcoRI, one with the restriction
endonuclease HindIII, and the third sample is uncut. The DNA
samples will be loaded into wells of an agarose gel and separated by
the process of electrophoresis. After migration of the DNA through an
electrical field, the gel will be stained with methylene blue, a dye
which binds to DNA.

When any molecule enters an electric field, the mobility or speed
at which it will move is influenced by the charge of the molecule,
the strength of the electrical field, the size and shape of the
molecule, and the density of the medium (gel) through which it is
migrating. When all molecules are positioned at a uniform starting
site on a gel and a gel is placed in a chamber containing a buffer
solution and electricity is applied, the molecules will migrate and
appear as bands. Nucleic acids, like DNA and RNA, move because of the
charged phosphate group in the backbone of the DNA molecule. Because
the phosphates are negatively charged at neutral pH, the DNA will
migrate through the gel toward the positive electrode.

In this exercise, we will use an agarose gel. In agarose,
the migration rate of linear fragments of DNA is inversely
proportional to their size; the smaller the DNA molecules, the faster
it migrates through the gel.

General Procedure

A: Preparing the Gel

1. Prepare the agarose gel for electrophoresis according to the
directions given by you teacher or in the kit.

2. Obtain the phage lambda DNA digested with EcoRI
endonuclease. The DNA is mixed with a gel-loading solution containing
a tracking dye, bromophenol blue, that will make it possible to
"track" the processes of its migration in the agarose gel.

3. Obtain the phage lambda DNA digested with HindIII
endonuclease. The Dna fragments are of a known size and will serve as
a "standard" for measuring the size of the EcoRI fragments
from step 2. It also contains tha tracking dye.

4. Obtain the undigested phage lambda DNA to use as a control. It
also contains the tracking dye.

B: Loading the Gel

Helpful Hints for Loading Gel

Pull a small amount of gel-loading solution into the end
of a micropipette. ( Do not allow

the solution to move up into the pipette, or bubbles will
be introduced into the well of the

1. Place the top on the electrophoresis
chamber and carefully connect the electrical leads to an approved
power supply (black to black and red to red). Set the voltage
to the appropriate level for your apparatus. When the current is
flowing, you should see bubbles on the electrodes.

2. Allow electrophoresis to proceed until
the tracking dye has moved nearly to the end of the gel.

3. After electrophoresis is complete,
turn off the power, disconnect the leads, and remove the cover
of the electrophoresis chamber.

D: Staining and
Visualization

Note: Wear Gloves

1. Carefully remove the gel bed from the
chamber and gently transfer the gel to a staining tray for staining.
Use the scooper provided with your kit or keep your hands under the
gel during the transfer. Do not stain in the electrophoresis
chamber.

E: Determining Fragment
Size

1. After observing the gel, carefully wrap
it in plastic wrap and smooth out all the wrinkles.

2. Using a marking pen, trace the outlines
of the sample wells and the location of the bands.

3. Remove the plastic wrap and flatten it
out on a white piece of paper on the laboratory bench. Save the gel
in a zip lock bag. Add several drops of buffer, store at 4degrees C.
You can make your measurments directly from the marks on the plastic
wrap.

Analysis and Results

Background Information

The size of the fragments produced by a specific endonuclease can
be determined by using standard fragments of known size. When you
plot the data on semilog graph paper, the size of the fragments is
expressed in the log of the number of base pairs they contain. This
allows data to be plotted on a straight line. The migration distance
of the unknown fragments, plotted on the x-axis, will allow their
size to be determined on the standard curve.

Graphing

A. Standard Curve for HindIII

1. Measure the migration distance(in cm) for each HindIII
band on your gel. Measure from the bottom of the sample well to
the bottom of the band. Measurment of the longest standard fragment
does not need to be measured (23,120 base pairs). Record these
measurments on table 6.1.

2. Plot the measured migration distance for each band of the
standard HindIII digest against the actual base pair (bp)
fragment sizes given in Table 6.1 using the semilog graph paper. Draw
the best fit line to your points. This will serve as a standard
curve.

B. Interpolated Calculations for EcoRI

From your standard curve for HindIII, made from known
fragment sizes, you can calculate fragment sizes resulting from a
digest with EcoRI. The procedure is as follows.

1. Measure the migration distance in cm for each EcoRI
band. Record the data in Table 6.1

2. Determine the sizes of fragments of lambda phage DNA digested
with EcoRI. Locate on the x axis the distance migrated by the
first EcoRI fragment. Using a ruler, draw a vertical line from
this point to its intersection with a best fit data line. Now extend
a horizontal line from intersection point to the Y axis. This point
gives the base pair size for this EcoRI fragment. Repeat this
procedure and determine the remaining EcoRI fragments. Enter
your interpolated data in Table 6.1, in the interpolated bp
column.

3. Your teacher will provide you with the actual bp data. Compare
your results to these actual sizes. Note: This interpolation
technique is not exact. You should expect as much as 10% to 15%
error.

2. Two small restriction fragments of nearly the same base-pair
size appear as a single band, even when the sample is run to the very
end of the gel. What could be dne to resolve the fragments? Why would
it work?